Endometrial Steroid Receptor Dysregulation and Its Association with Vitamin D, AMH, and Inflammation in Recurrent Implantation Failure: A Case-Control Study.

Implantation is a highly regulated biological process in which the blastocyst adheres to the endometrium and initiates epithelial invasion, ultimately leading to placenta development. Successful reproduction requires precise temporal coordination between blastocyst maturation and endometrial differentiation, which occurs within a defined interval known as the window of implantation (WOI) ( 1 , 2 ). Although multiple factors contribute to this process, the molecular mechanisms governing the interaction between endometrial secretory activity and the blastocyst remain incompletely understood ( 3 ). The establishment of successful implantation depends on endometrial receptivity, a crucial determinant of fe male fertility ( 4 ). Impaired receptivity can disrupt the implantation process, contributing to infertility. This process is governed by a complex interplay of molecular signals, including growth factors, hormones, cytokines and adhesion molecules that facilitate the embryo-maternal interface ( 3 , 5 ). Throughout the menstrual cycle, ovarian steroids regulate uterine receptivity: during the preovulatory phase, estrogen (E2) promotes endometrial proliferation, while progesterone (P4) induces secretory transformation of the estrogen-primed endometrium. The actions of these hormones are mediated by their respective receptors , which are expressed in both stromal and epithelial cells of the endometrium ( 3 ). The presence of P4 following E2 priming is essential for initiating the molecular events required for implantation during the mid-secretory phase of the menstrual cycle. This hormonal interplay regulates the WOI and activates downstream signaling pathways ( 5 ). The endometrium expresses estrogen receptors ( ESRs ) and progesterone receptors ( PGRs ) in both the epithelial and stromal compartments. Activation of PGRs by P4 regulates the expression of genes critical for implantation ( 3 , 5 ). During the proliferative phase, ESR-α expression in crease in response to E2, but declines during WOI under the influence of P4. This downregulation of ESR-α is essential for the establishment of endometrial receptivity. The transition is accompanied by the expression of several genes such as of PGR-B, PGR-A, ESR-2, Beta 3 Integrin ( ITGB3 ), and Glycodelin-A ( GdA ) particu larly during the mid-luteal phase ( 6 ). Women with en dometriosis and polycystic ovarian syndrome (PCOS) often exhibit abnormally elevated ESR-α levels during the implantation window, which has been linked to a reduction in in ITGB3 expression, a recognized marker of endometrial receptivity ( 7 ). Recent studies have further clarified the interplay between E2 and anti-Mülle rian hormone (AMH) during puberty and gonadotropin recovery. E2 suppresses AMH expression via ESR-α binding, whereas ESR-β binding enhances AMH expression. Moreover, both E2 and its receptors regulate AMH gene expression through the estrogen response element (ERE) within the AMH promoter region ( 8 , 9 ). Furthermore, PGR expression is significantly reduced in the perivascular regions of women with RIF compared to healthy controls ( 10 ). In these patients, the decreased expression of ESR-β and PGR-B in vascular and perivascular cells may reflect impaired angiogenesis and decidualization, potentially contributing to implantation failure. Endometrial endothelial cells express ESR-β throughout the menstrual cycle, where it is thought to play a role in regulating the vascular and angiogenic changes required for early placentation and implantation ( 10 ). Vitamin D3 (Vit-D3) is essential for regulating the immune system and reducing inflammation, with effects on progesterone (P4) function during pregnancy through the enhancement of membrane progesterone receptor (mPR) gene expression in CD4+ T cells ( 11 , 12 ). Vit-D3 supplementation influences post-translational modifications and PGR isoforms, affecting their stability and activity, which is crucial for endometrial tissue function ( 13 ). In a rat model, Vit-D3 deficiency was linked to impaired P4 receptor functionality and decreased expression of key genes, such as HOXA-10 and FKBP52, ultimately affecting uterine receptivity during implantation ( 14 ). Patients with recurrent implantation failure (RIF) and low Vit-D3 levels showed significant downregulation of genes associated with decidualization and implantation compared to those with sufficient Vit-D3, highlighting its importance in embryo implantation ( 12 , 15 , 16 ). Additionally, elevated high-sensitivity C-reactive protein (hs-CRP), a marker of inflammation, has been associated with reduced fecundability and poorer outcomes in assisted reproductive technology (ART). Studies have indicated that higher hs-CRP levels in women undergoing in vitro fertilization (IVF) correlate with lower clinical pregnancy and live birth rates ( 17 ). A recent study indicated that E2 acts as a more potent pro-inflammatory sex steroid compared to P4 ( 18 ). P4 is well recognized for its immunomodulatory functions, which are critical in suppressing pro-inflammatory and cytotoxic T-cell responses during pregnancy ( 19 ). Furthermore, a mouse study demonstrated that P4 and its receptor (PGR) regulate the endometrial inflammatory response by modulating the expression of the nuclear factor kappa B inhibitor alpha ( NFKBIA ) gene. Knockdown of PGR-B and ESR-β has been shown to increase inflammation by upregulating prostaglandin endoperoxide synthase 2 ( Ptgs2 ) gene expression and elevating prostaglandin E2 levels ( 20 ). The chicken ovalbumin upstream promoter-transcription factor II ( COUP-TFII ) gene and protein, which are activated by the P4-PGR-B complex, play key roles in regulating inflammatory cytokines, embryo implantation, and endometrial disorders such as endometriosis. In murine models, COUP-TFII deficiency results in E2 dominance, infertility, and increased inflammation. Similarly, reduced COUP-TFII gene expression in endometriosis contributes to P4 resistance, promoting inflammation and cellular proliferation ( 21 , 22 ). Recent evidence suggests that RIF may result from disruptions in the expression of implantation-related genes, protein levels, and hormonal signaling. Based on these insights, we hypothesize that overexpression of ESR-α coupled with reduced expression of PGR-B may impair the development of endometrial receptivity. Therefore, the primary aim of this study was to evaluate the expression levels of ESR-α, ESR-β , and PGR-B in patients with RIF, and to investigate potential associations between steroid receptor expression and markers such as Vit-D3 status, AMH, and hs-CRP as indicators of inflammation.

Table 1 summarizes the clinical characteristics of patients with RIF patients and controls. There were no significant differences between the two groups in terms of age and body mass index (BMI). Similarly, the under lying cause of infertility did not differ significantly between groups. Both cohorts exhibited comparable mean numbers of retrieved oocytes and embryos. However, the implantation rate was significantly lower in the RIF group (4%) compared to controls (30%). Anthropometric and biochemical characteristics RIF; Recurrent implantation failure, BMI; Body mass index, AMH; Anti-müllerian hormone, Vit-D3; Vitamin D3, P4; Progesterone, E2; Estrogen, hs-CRP; High-sensitive C reactive protein, 1; Independent sample t test, 2; Mann-Whitney U test, and *; P=0.0001. Table 1 presents the hormonal profiles of the study groups. No significant differences were observed between RIF patients and controls in terms of serum concentrations of P4 and E2. However, significant differences were detected in AMH, Vit-D3, and hs-CRP levels between the groups. Specifically, AMH levels were significantly lower in the RIF group compared to fertile controls (median: 1.60 ng/ml; range: 0.8-5.59 ng/ml vs. 4.30 ng/ ml; range: 1.10-8.70 ng/ml, P=0.001). In contrast, hs CRP levels were significantly elevated in RIF patients (median: 3.9 mg/L; range: 2.57-6.9 mg/L) relative to controls (median: 1.1 mg/L; range: 0.4-3.24 mg/L) (P=0.001). Additionally, Vit-D3 levels were markedly reduced in the RIF group (median: 25.6 ng/mL; 10.5 62.9 ng/mL) compared to controls (median: 56.5 ng/ mL; range: 15.9-127.8 ng/mL, P=0.001). Figure 1A-C presents box-and-whisker plots illustrating biomarker distributions in both groups. Comprehensive clinical and biochemical characteristics are detailed in Table 1. As illustrated in Figure 1D-F, endometrial expression of ESR-α gene was significantly upregulated in patients with RIF compared to fertile controls (P=0.001). Conversely, expression levels of ESR-β and PGR-B genes were significantly downregulated in the RIF group relative to the control group (P=0.001). In patients with RIF, endometrial expression of ESR-α was significantly and negatively correlated with the expression of ESR-β and PGR-B genes (P=0.001 and P= 0.0001, Fig 2A, B ). Conversely, a significant positive correlation was observed between PGR-B and ESR-β expression levels (P=0.005, Fig .2C ). Additionally, in women with RIF, serum hs-CRP levels were inversely correlated with AMH and Vit-D3 concentrations, while a significant positive correlation existed between AMH and Vit-D3 levels (P=0.001, P=0.01, P=0.001, Fig .2D-F ). A significant negative correlation was observed between the endometrial expression of the ESR-α gene and serum AMH levels (P=0.0001). In contrast, ESR-α expression was positively associated with hs-CRP levels (P=0.001, Fig .3A, B ). Furthermore, the endometrial expression levels of ESR-β and PGR-B were positively correlated with AMH concentrations (P=0.001 and P=0.001, Fig .3C, D ). Conversely, a significant negative correlation was detected between PGR-B expression and hs-CRP levels in RIF patients (P=0.010, Fig .3E ). In fertile women without RIF, a significant positive correlation was observed between the endometrial expression levels of PGR-B and ESR-β (P=0.010, Fig .4A ). Additionally, PGR-B expression showed significant negative correlation with hs-CRP levels ( Fig .4B ) and with serum Vit-D3 concentrations (P=0.001, P=0.0001, Fig .4C ). Moreover, Vit-D3 levels were significantly correlated with both hs-CRP (r=-0.297, P=0.050) and AMH levels (r=0.314, P=0.050) in fertile women ( Fig .4D-E ). Serum concentrations of AMH, Vit-D3 and hs-CRP, along with endometrial expression levels of ESR-α, ESR-ϐ and PGR-B genes in women with RIF and fertile controls. Statistical analysis: A-C. Independent samples t test was used for panels, and D-F. Mann–Whitney U test was applied for panels. Values presented as mean and range (minimum-maximum). ****; P<0.0001, AMH; Anti-Müllerian hormone, Vit-D3; Vitamin D3, hs-CRP; High-sensitive C-reactive protein, ESR-α; Estrogen receptor-α, PGR-B; Progesterone receptor-B, ESR-β; Estrogen Receptor-β, and RIF; Recurrent implantation failure. Significant correlations between endometrial expression levels of steroid hormone receptor genes and circulating biomarkers in patients with RIF. A-C. These panels show correlations among ESR-α, ESR-ϐ, and PGR-B gene expression levels. D-F. These panels illustrate correlations between serum levels of hs-CRP, AMH, and Vit-D3. Statistical analysis: Spearman’s rank correlation coefficient was used. RIF; Recurrent implantation failure, hs-CRP; High sensitive C-reactive protein, AMH; Anti-Müllerian hormone, ESR-α; Estrogen receptor-α, ESR-β; Estrogen receptor-β, PGR-B; Progesterone receptor-B, and Vit-D3; Vitamin D3. Significant correlations between endometrial expression levels of ESR-ϐ and PGR-B genes and serum concentrations of AHM and hs-CRP biomark ers in patients with RIF. Statistical analysis was performed using Spearman’s rank correlation coefficient. AMH; Anti-Müllerian hormone, hs-CRP; High sensitive C-reactive protein, PGR-B; Progesterone receptor-B, ESR-β; Estrogen receptor-β, Vit-D3; Vitamin D3, and RIF; Recurrent implantation failure. The ROC curve analysis demonstrated excellent discrimi natory performance for endometrial gene expression levels. The area under the curve (AUC) for ESR-α was 1.00, [95% confidence interval (CI): 1.00-1.00), P=0.001], for ESR-β was 0.988, [95% CI: 0.969-1.00, P=0.001] and for PGR-B was 0.898, [95% CI: 0.841-0.955, P=0.001], indicating strong discriminative ability between RIF patients and fertile women. Similarly, serum biomarker analysis revealed robust performance: AMH [AUC: 0.876; 95% CI: 0.8075 0.9457, P=0.001], Vit-D3 [AUC: 0.820, 95% CI: 0.7370 0.9042, P=0.001] and hs-CRP [AUC: 0.994, 95% CI: 0.9846-1.000, P=0.001] ( Fig .5A-F ). These findings support the potential diagnostic value of both endometrial gene expression and serum biomarkers in distinguishing RIF patients from fertile controls. Significant correlations between endometrial expression levels of ESR-α, ESR-β and PGR-B genes and serum concentrations of relevant biomarkers in fertile women without RIF. Statistical analysis was performed using Spearman's rank correlation coefficient. RIF; Recurrent implantation failure, hs-CRP; High-sensitive C-reactive protein, Vit-D3; Vitamin D3, PGR-B; Progesterone receptor-B, ESR-β; Estrogen receptor-β, and AMH; Anti-Müllerian hormone. The ROC curve effectively evaluates the ability of biomarkers to distinguish recurrent implantation failure cases from controls. ROC curve analysis of endometrial gene expression levels of A. ESR-α, B. ESR-β and C. PGR-B and serum concentrations of D. AMH, E. Vit-D3, and F. hs-CRP in patients with RIF. AUC; Area under the curve, ROC; Receiver operator characteristic, AMH; Anti-Müllerian hormone, Vit-D3; Vitamin D3, hs-CRP; High-sensitive C-reactive protein, ESR-α; Estrogen receptor-α, ESR-β; Estrogen receptor-β, PGR-B; Progesterone receptor-B, and RIF; Recurrent implantation failure Infertility can result from implantation failure, which largely depends on proper endometrial receptivity. This receptivity is established through finely regulated in teractions between E2 and P4 signaling pathways. Disruptions in the expression of key genes such as ESR-α, ESR-β , and PGR-B have been implicated in recurrent reproductive failure (RRF), potentially affecting serum levels of AMH and inflammation markers ( 24 ). The present study aimed to evaluate the endometrial expression levels of ESR-α, ESR-β, and PGR-B in pa tients with RIF and to investigate potential correlations between gene expression and relevant biochemical parameters in comparison with fertile controls. The present study found that ESR-α gene expression was significantly upregulated in endometrial tissue of RIF patients compared to fertile women. In contrast, ESR-β expression was markedly lower in women with RIF women than in fertile controls. These findings are consistent with those reported by Dorostghoal et al. ( 6 ), who observed significant overexpression of ESR-α in the mid-luteal endometrium of women with unexplained infertility (UI) compared to fertile women. Similarly, increased ESR-α expression has also been reported in peripheral blood mononuclear cells (PBMC) of women with UI infertility compared to healthy individuals ( 25 ). Lessey et al. ( 22 ) suggested that overex pression of ESR-α leads to a downregulation of the β3 integrin ( ITGB3 ) gene and its corresponding protein. ITGB3 plays a vital role in embryo implantation, particularly during the mid-secretory phase of the menstrual cycle. Consequently, reduced β3 integrin gene expression in women with endometriosis may contribute to infertility ( 22 ). A key finding of our study was the significant deregulation of PGR-B gene in women with RIF compared to fertile controls. Although serum levels of E2 and P4 were comparable between the groups, P4 concentrations tended to decrease during the mid-luteal phase in RIF patients. Recent studies suggest that downregulation of the PGR-B may be linked to diminished P4 receptor expression or secondary P4 resistance, both of which can contribute to RIF ( 24 , 26 ). We found that patients with RIF exhibited significant ly higher levels of hs-CRP compared to fertile women without RIF. Moreover, hs-CRP levels were positively correlated with ESR-α gene overexpression in RIF patients. In contrast, hs-CRP levels showed an inverse correlation with endometrial expression of PGR-B and ESR-β genes in both RIF and fertile women. Supporting our findings, Zhang et al. ( 17 ) demonstrated that hs-CRP serves as a biomarker of inflammation in women undergoing IVF and is closely associated with clinical pregnancy outcomes, live birth, and implantation failure ( 17 , 27 ). It is important to recognize the critical role of PGR in resolving ovulatory inflammation by suppressing LH-induced PTGS2 gene expression, thus protecting the ovary from damage caused by repeated inflammatory cycles ( 20 ). Consistent with this, our results showed a significant correlation between PGR-B gene expression and hs-CRP levels in both studied groups, highlighting the potential role of PGR-B in mitigating inflammation and modulating immune response. Furthermore, AbdulHussain et al. ( 28 ) reported that progesterone supplementation can modulate the immune system by suppressing the production of inflammatory cytokines such as IL-2, TNF-α, INF-γ, and IL-17a in women with a history of unexplained recurrent spontaneous miscarriage (uRSM) ( 28 , 29 ). Consistent with previous studies ( 20 , 28 , 29 ), our findings demonstrate that elevated hs-CRP in patients with RIF could serve as a valuable biomarker of inflammation. This increase correlates with overexpression of ESR-α and decreased expression of PGR-B. These observations suggest a potential interplay between dysregulated steroid hormone signaling and a pro-inflammatory environment within the endometrium, which may contribute to the pathophysiology of RIF. Our results revealed that serum levels of Vit-D3 were significantly lower in RIF patients compared to fertile women. In RIF patients, Vit-D3 levels were inversely correlated with hs-CRP levels, suggesting a potential link between Vit-D3 deficiency and systemic inflammation. Additionally, in fertile women, a strong positive correlation was observed between Vit-D3 levels and endometrial PGR-B gene expression. Vitamin D3 plays a crucial role in regulating immune and inflammatory responses, and numerous studies have linked low Vit-D3 levels to increased inflammation in RIF patients. Expression of the Vitamin D receptor (VDR) has been identified in the endometrium, fallopian tube epithelial cells, granulosa cells, and cumulus oophorus cells of the ovary ( 14 ). Recent research further demonstrated that RIF patients with Vitamin D3 deficiency show significant downregulation of key genes involved in decidualization and implantation—including VDR, PGR, PRL, IGFBP1, and HOXA10- compared to RIF patients with adequate Vitamin D3 levels ( 30 ). An animal study by Ashour et al. ( 14 ) demonstrated that Vit-D3 supplementation is associated with increased expression of E2 and P4 receptors as well as the FKBP52 gene in mammals ( 13 , 14 ). Similarly, Hosseinirad et al. ( 13 ) reported that Vit-D3 intake sig nificantly upregulated PGR gene expression and protein levels, including the phosphorylated form of PGR protein, in endometrial stromal cells (eSC). Additional research has shown that Vit-D3 influences the uterus throughout the estrous cycle in pigs, with the highest expression of VDR protein occurring in the myometrium. While Vit-D3 supplementation did not affect uterine P4 release, it significantly increased E2 release in the myometrium ( 31 ). Moreover, insufficient levels Vit-D3 are commonly observed in women undergoing assisted reproductive treatments and have been associated with reduced live birth rates ( 32 ). Our findings are consistent with these studies, supporting the notion that Vit-D3 plays a crucial role in embryo implantation and endometrial cell function by upregulating PGR-B gene and other implantation-related factors. Further research is warranted to elucidate the molecular mechanisms underlying of Vitamin D3's regulatory effects on PGR gene expression and protein activity. Our recent study found that AMH levels were significantly lower in patients with RIF compared to fertile women. We also observed a positive and significant correlation between endometrial expression levels of ESR-β and PGR-B with AMH levels, whereas a significant negative correlation existed between ESR-α expression and AMH levels. A recent study by Tanimoto et al. ( 33 ) demonstrated that elevated E2 levels and overexpression of ESR-α could interact with the AMH gene via estrogen response elements (ERE), resulting in increased expression of AMH and AMHRII gene in the ovaries. This interaction may disrupt normal follicle development, leading to abnormal follicle formation ( 9 , 33 , 34 ). Additionally, Grynberg et al. ( 9 ) showed that estradiol, acting through ESR-α , could suppress AMH mRNA expression in human granulosa cells stimulated with hCG. They also reported that a GnRH antagonist and estradiol benzoate decreased AMH expression in certain pre-antral and small antral follicles in pre-pubertal rats. These findings highlight a complex regulatory relationship between steroid receptors and AMH gene expression during folliculogenesis. In a mouse model study, the absence of PGR and ESR-β genes led to reduced AMH expression in ovulatory granulosa cells, highlighting the critical role of PGR-B and ESR-β in decidulization and endometrial receptivity ( 20 ). In women with severe preeclampsia (sPE), an imbalance in ESR-α and PGR-B gene expression-particularly a marked reduction in PGR-B—has been associated with defective decidualization (DD) and disruption of key cellular signaling pathways ( 35 ). Furthermore, Al-Lamee et al. ( 10 ) reported that women with recurrent early pregnancy loss (RLEP), recurrent fetal loss (RFL), and RIF exhibited downregulated expression of the ESR-β and PGR-B in the glandular epithelium, perivascular cells, and vascular endothelium compared to healthy controls. Our ROC curve analysis demonstrated that circulat ing AMH, Vit-D3, hs-CRP, and endometrial steroid receptors exhibit excellent discriminatory power for distinguishing RIF from other pregnancy-related disorders. For example, Alson et al. ( 36 ) reported an AUC of 0.56 for AMH in women undergoing IVF, which is notably lower than the AUC observed in our study (0.876). Similarly, Hou et al. ( 37 ) reported AUC val ues of 0.664 and 0.661 for AMH in predicting cumulative live birth rate and cumulative clinical pregnancy rate, respectively, indicating that higher AMH levels are associated with increased likelihood of successful implantation and live birth. Consistent with these findings, our results suggest that AMH and the PGR-B and ESR genes may serve as reliable predictors of successful IVF outcomes and help reduce the risk of RIF. While our study provides valuable insights into the role of endometrial PGR-B, ESR-β, and ESR-α in fertility and IVF success, several limitations should be ac knowledged. First, the sample size of UI cases was relatively small, which may limit the generalizability of our findings. Second, protein expression levels were not assessed using immunohistochemistry or western blotting, techniques that could provide a more detailed understanding of the molecular mechanisms involved. Third, the study did not include measurements of cytokines levels or inflammatory and anti-inflammatory gene expression in serum or endometrial tissue, restricting our understanding of the immune environment influencing endometrial receptivity. Addressing these gaps in future research may help identify additional biomarkers and facilitate the development of novel therapeutic strategies for treating recurrent implantation failure.

The study demonstrated that endometrial ESR-β and PGR-B gene expression were significantly downregulated , while ESR-α expression was markedly upregulated in women with RIF. In addition, RIF patients exhibited lower serum levels of AMH and Vit-D3, alongside elevated hs-CRP levels, compared to fertile controls. Notably, AMH and hs-CRP levels were significantly associated with the aberrant expression of ESR-α, suggesting their potential involvement in the pathophysiol ogy of RIF These findings highlight the diagnostic and predictive value of AMH levels and PGR-B/ESRs gene expression profiles in assessing IVF outcomes and the risk of RIF. Further studies are warranted to elucidate the underlying molecular mechanisms governing endometrial receptivity and implantation failure, which may pave the way for the development of targeted therapeutic strategies.

This case-control study was conducted between June 2021 and December 2023, following approval by the Ethics Committee of Iran University of Medical Sciences (IR. IUMS.FMD.REC.1402.040). Written informed consent was obtained from all participants prior to enrollment. A total of 100 women were included, comprising 50 patients diagnosed with recurrent implantation failure (RIF) and 50 age-matched fertile controls. The sample size was calculated using the Chi-square test, assuming a statistical power of 80% and a significance level of 0.05. This case-control study included 50 female patients under the age of 42 who were referred to the RIF Medical Center at Arash Hospital (Tehran, Iran). All patients had undergone IVF or intracytoplasmic sperm injection (ICSI) treatment and experienced at least three failed embryo transfers, despite receiving four or more high-grade, morphologically normal embryos. The control group consisted of 50 women with no prior history of IVF, who presented with secondary infertility due to male factor, tubal factor, or unexplained causes. All control participants had a history of at least one successful full-term pregnancy resulting in a live birth. Participants were included if they had regular menstrual cycles (28-32 days) and normal endocrine profiles. Exclusion criteria for both patient and control groups were: poor embryo quality or poor ovarian response; known uterine or endometrial pathologies; hereditary or acquired thrombophilia; diabetes mellitus; thyroid disorders; poly cystic ovary syndrome (PCOS); intrauterine abnormali ties; adenomyosis; endometriosis; positive anti-lupus an ticoagulant; abnormal chromosomal karyotypes; history of miscarriage; endocrine disorders; infectious diseases; and use of contraceptive agents. Both female and male partners were screened for these conditions. All participants in both groups were required to meet inclusion criteria of regular ovulation cycles (28-32 days) and normal endocrine profiles. Ovulation timing was determined through daily morning serum measurements of luteinizing hormone (LH) levels, using a quantitative ELISA kit (Catalog No. PT LH-96, Pistazteb, Tehran, Iran) in strict accordance with the manufacturer’s protocol. Transvaginal ultrasonogra phy was performed with the PHILIPS Affiniti 70 ultra sound system (Philips Healthcare, Bothell, WA, USA), equipped with an endovaginal transducer (Philips C10-3v probe, 3 -10 MHz). The LH surge was designated as LH 0, and the implantation window was defined as LH+7. Endometrial biopsies were obtained accordingly on LH+7 via hysteroscopy in RIF patients and using a Pipelle catheter in control subjects. Hysteroscopy enabled both sample collection and the detection of intrauterine abnormalities that might not be identified through imaging modalities. Peripheral blood samples were collected simultaneously for serum analysis. All biological specimens were imme diately transported to the laboratory and stored at -80°C for subsequent total RNA extraction. All participants underwent standard fertility evaluations and were deemed suitable candidates for IVF or ICSI. Ovarian stimulation was performed using either recombinant follicle-stimulating hormone (Cinnal-F, CinnaGen; Iran) or human menopausal gonadotropin (HMG, Menotropin, Poish Darou, Iran). Premature LH surges was prevented with a gonadotropin-releasing hormone (GnRH) antagonist (Cetronax, Ronak, Iran). Final oocyte maturation was induced with 10,000 IU of human chorionic gonadotropin (hCG, Gonarx, Ronak, Iran), and oocyte retrieval was carried out 36 hours later under ultrasound guidance. One or two fresh grade A blastocyst were transferred using an embryo transfer catheter (Cat. No: BR344128, Rada, Behrad, Iran) preloaded with embryoculture medium that had been equilibrated under sterile conditions at 37°C to minimize exposure to ambient air and light. Luteal phase support was provided with 800 mg/day of intravaginal P4 (Fertigest, Aburaihan, Iran). During the mid-luteal phase of the implantation window (approximately 5-7 days after the LH surge, or LH+7), peripheral blood and endometrial tissue samples were collected from all participants within the same menstrual cycle, prior to embryo transfer. A 5 mL peripheral blood sample was drawn under sterile conditions via venipuncture using a vacuum collection system (Cat. No. EN-14820, Ava Pezeshk, Tehran, Iran). After clotting for 15 minutes at room temperature, samples were centrifuged at 1000 rpm for 15 minutes. The re sulting serum was aliquoted into RNase-free microtubes (Cat. No. AM12425, Thermo Fisher Scientific, Waltham, Massachusetts, USA) and stored at -70°C for subsequent analyses. Endometrial tissue sampling was performed on the same day: hysteroscopy was used for the RIF group, and a Pipelle catheter (Cat. No: BR344128, Rada, Behrad, Iran), was used for the control group, in accordance with established protocols ( 23 ). Hysteroscopy also allowed for the identification of intrauterine abnormalities that may not be detectable by other imaging techniques. Serum levels of the AMH, E2, and P4 were measured using ELISA kits (AMH: Cat. No. PT-AMH-96 Pishtazteb, E2: Cat. No. 4925-300A and P4: Cat. No. 2734-96 all from Monokit, Tehran, Iran). In addition to hormonal analysis, two non-hormonal biomarkers-hs CRP and vitamin D3 (25-hydroxyvitamin D)-were also quantified using specific ELISA kits (hs-CRP: Cat. No.0234-96, Monokit, Vit-D3: Cat. No. PT-Vit D-96, Pishtaz Teb, Tehran, Iran), according to the manufacturer’s protocols. Total RNA was extracted from endometrial tissue samples of both patients and controls using an RNA extraction kit according to the manufacturer’s protocol (Cat. No. FABRK001, Yekta Tajhiz- Tehran, Iran). The concentration and purity of the extracted RNA were assessed using a NanoDrop One spectrophotometer (ND-ONE-W, Thermo Scientific, USA) and RNA in tegrity was confirmed via 3% agarose gel electropho resis (Cat. No: 161-0722, Bio-Rad, USA) complementary DNA (cDNA) was synthesized from 1 µg of total RNA using a reverse transcription kit (cDNA synthesis premix, Zist Virayesh, Cat. No: ZV06030050, Zist Virayesh, Tehran, Iran). Quantitative real-time PCR (qRT-PCR) was performed to evaluate the expression levels of ESR-α, ESR-β, and PGR-B genes. The reactions were conducted using a SYBR Green Master Mix (Cat. No. FABRK001, Yekta Tajhiz Azma,Tehran, Iran) on a StepOne Real-Time PCR System Applied Biosystems, USA). Thermal cycling conditions included an initial denaturation at 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds and annealing/extension at 60°C for 40 seconds. Gene expression levels were calculated using the 2-ΔΔCt method and normalized to GAPDH as the internal reference gene ( 23 ). The applied primers were used as below: 5′-TCTACCCGCCCTATCTCAACTACC-3′ R: 5′-TGTGCTGCCCTTCCATTGCC-3′; 5′-CGATGATGGGCTTACTGACC-3′ R: 5′-CCCTCTACACATTTTCCCTGG-3′; 5′-ATGAGGGGAAATGCGTAGAAG-3′ R: 5′-ATCACCCAAACCAAAGCATCG-3′; 5'-CCCCTTCATTGACCTCAACTAC- 3' R: 5'-GATGACAAGCTTCCCGTTCTC-3′ Data analysis was performed using SPSS software version 16 (IBM Corp., USA). Nonparametric variables are presented as median values with ranges (minimum to maximum). Continuous variables were compared us ing the Student’s t test for normally distributed data or the Mann-Whitney U test for non-normally distributed (skewed) data. Categorical variables were analyzed us ing the Chi-square (χ²) test. Skewed variables were log transformed using natural logarithms prior to analysis. To compare the expression levels of ESR-α, ESR-β, PGR-B genes and serum levels of AMH, P4, E2, Vit-D3, and hs- CRP between groups, the Mann-Whitney U test was applied. Spearman’s correlation coefficient was used to assess relationships between variables. Whisker plots were generated with GraphPad Prism version 8.0.0 for Windows (GraphPad Software, USA). A P<0.05 was considered statistically significant.